Methods and compositions for administration of 3-halopyruvate and related compounds for the treatment of cancer

ABSTRACT

The present disclosure relates to the discovery that compounds of the invention, particularly 3-bromopyruvate and related compounds, can be safely administered at concentrations effective for the treatment of cancer when formulated with an acidity of greater than or equal to pH of 2 and less than or equal to a pH of 6. Disclosed herein are novel and improved methods and compositions for the treatment of cancer using 3-halopyruvate and related compounds.

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e)to Provisional Application Ser. No. 61/165,239, filed Mar. 31, 2009,Provisional Application Ser. No. 61/097,408, filed Sep. 16, 2008 andProvisional Application Ser. No. 61/090,793, filed Aug. 21, 2008, thecontents of which are all incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The knowledge that cancer cells rely on increased glycolysis rather thanoxidative phosphorylation for survival is known as “The WarburgHypothesis” (Warburg, O., Science, 123:309-314 (1956)). This conceptconstitutes the basis for using glycolysis and its associated enzymes asunique targets for the development of new anticancer therapeutic agents(Shaw, R. J., Curr. Opin. Cell Biol., 18:598-608 (2006); Gatenby, R. A.and Gillies, R. J., J. Biochem. Cell Biol., 39:1358-1366 (2007)). Onesuch agent is 3-bromopyruvate (3-BrPA), a synthetic brominatedderivative of pyruvic acid that acts as an irreversible glycolyticinhibitor (Ko et al., Cancer Lett., 173:83-91 (2001); Geschwind et al.,Cancer Res., 62:3909-3913 (2002)). Early studies demonstrated that3-BrPA is able to completely eradicate tumors implanted in rabbit liverswhen administered directly into the liver by intra-arterial injection,resulting in a significant survival benefit in advanced stages of thedisease. The therapeutic dose was found to be to be 1.75 mM in 25 ml ofphosphate-buffered saline (PBS), when given as a continuous, 1 hourintra-arterial infusion (Vali et al., J. Vasc. Interv. Radiol.,18:95-101 (2007)). Furthermore, when the animals were treated at arelatively early stage of their cancer, effective local control of thetumor resulted in the achievement of complete remission of the cancer.In addition to its excellent therapeutic profile, intra-arteriallydelivered 3-BrPA also had a favorable biodistribution profile with ahigh tumor uptake and no negative effects on healthy tissue (Vali etal., The Journal of Pharmacology and Experimental Therapeutics,327(1):32-7 (2008)).

These results highlight that 3-BrPA is a highly promising anti-canceragent. However, a major limitation for future clinical use of thiscompound is the fact that the preclinical testing was conducted using arather uncommon delivery method for anti-cancer agents, i.e.intra-arterially directly into the liver. Because 3-BrPA is anon-specific alkylating agent, it is thought to be a highly toxiccompound and it was believed that the intra-arterial approach wasrequired to prevent systemic toxicities (Chang et al., Acad. Radiol.,14(1)85-92 (2007)).

Because intra-arterial administration is a technically challengingmethod for delivery an anti-cancer agent and is not applicable for everytype of cancer, other 3-BrPA delivery methods are greatly desired.

SUMMARY

The present invention relates to novel methods and compositions for theadministration of 3-halopyruvate and related anti-cancer compounds. Theinvention is based at least in part on the observation that safeadministration of an effective concentration of 3-halopyruvate or3-halopyruvate related compounds could be achieved if the compound wasadministered in a formulation with a pH of between 2 and 6.

In some embodiments, the invention relates to a method of treatingcancer in a subject that includes administering to the subject aneffective amount of a pharmaceutical composition that contains apharmaceutical agent represented by the general formula:

wherein, independently of each occurrence:

-   -   X represents a halide, a sulfonate, a carboxylate, an alkoxide,        or an amine oxide;    -   R₁ represents OR, H, N(R″)₂, C1-C6 alkyl, C6-C12 aryl, C1-C6        heteroalkyl, or C6-C12 heteroaryl;    -   R″ represents H, C1-C6 alkyl, or C6-C12 aryl;    -   R represents H, alkali metal, C1-C6 alkyl, C6-C12 aryl or        C(O)R′; and    -   R′ represents H, C1-C20 alkyl or C6-C12 aryl;        and wherein the pharmaceutical composition has an acidity of        greater than or equal to about pH 2 and less than or equal to        about pH 6. In some embodiments the acidity of the        pharmaceutical composition is greater than or equal to about pH        3 and less than or equal to about pH 5. In certain embodiments        the acidity of the pharmaceutical composition is pH 4.

In some embodiments of the invention, the pharmaceutical agent is3-halopyruvate. In certain embodiments the pharmaceutical agent is3-bromopyruvate.

In certain embodiments, the pharmaceutical composition is administeredorally. In some embodiments the pharmaceutical agent is administered ata dose of greater than or equal to about 150 milligrams per kilogram ofbody weight and less than or equal to about 250 milligrams per kilogramof body weight.

In some embodiments of the invention, the pharmaceutical compositionfurther includes NaHCO₃. In certain embodiments, the molar concentrationof the pharmaceutical agent is within 5 fold or 2 fold of the molarconcentration of NaHCO₃. In some embodiments the molar concentration ofthe pharmaceutical agent is about equal to the molar concentration ofNaHCO₃. In some embodiments, the molar concentration of thepharmaceutical agent is less than about 2 M. In certain embodiments, themolar concentration of the pharmaceutical agent is about 1 M. In someembodiments the concentration of the pharmaceutical agent is less thanor equal to about 0.03 milligrams per milliliter.

In some embodiments, the methods of the invention further include theadministration of a chemotherapeutic agent. In some instances,chemotherapeutic agent is altretamine, asparaginase, BCG, bleomycinsulfate, busulfan, camptothecin, carboplatin, carmusine, chlorambucil,cisplatin, claladribine, 2-chlorodeoxyadenosine, cyclophosphamide,cytarabine, dacarbazine imidazole carboxamide, dactinomycin,daunorubicin-dunomycin, dexamethosone, doxurubicin, etoposide,floxuridine, fluorouracil, fluoxymesterone, flutamide, fludarabine,goserelin, hydroxyurea, idarubicin HCL, ifosfamide, interferon alfa,interferon alfa 2a, interferon alfa 2b, interfereon alfa n3, irinotecan,leucovorin calcium, leuprolide, levamisole, lomustine, megestrol,melphalan, L-sarcosylin, melphalan hydrochloride, MESNA,mechlorethamine, methotrexate, mitomycin, mitoxantrone, mercaptopurine,paclitaxel, plicamycin, prednisone, procarbazine, streptozocin,tamoxifen, 6-thioguanine, thiotepa, topotecan, vinblastine, vincristineor vinorelbine tartrate.

In some embodiments, the invention relates to a method of treating asolid tumor. In certain embodiments the invention relates to thetreatment of liver cancer, pancreatic cancer, lung cancer, or breastcancer.

Certain aspects of the invention relate to the pharmaceuticalcomposition that contains a pharmaceutical agent represented in thegeneral formula:

wherein, independently of each occurrence:

-   -   X represents a halide, a sulfonate, a carboxylate, an alkoxide,        or an amine oxide;    -   R₁ represents OR, H, N(R″)₂, C1-C6 alkyl, C6-C12 aryl, C1-C6        heteroalkyl, or C6-C12 heteroaryl;    -   R″ represents H, C1-C6 alkyl, or C6-C12 aryl;    -   R represents H, alkali metal, C1-C6 alkyl, C6-C12 aryl or        C(O)R′; and    -   R′ represents H, C1-C20 alkyl or C6-C12 aryl;        and wherein the pharmaceutical composition has an acidity of        greater than or equal to about pH 2 and less than or equal to        about pH 6. In some embodiments the acidity of the        pharmaceutical composition is greater than or equal to about pH        3 and less than or equal to about pH 5. In certain embodiments        the acidity of the pharmaceutical composition is pH 4.

In some embodiments of the invention, the pharmaceutical agent is3-halopyruvate. In certain embodiments the pharmaceutical agent is3-bromopyruvate.

In certain embodiments the pharmaceutical composition is formulated fororal administration.

In some embodiments of the invention, the pharmaceutical compositionfurther includes NaHCO₃. In certain embodiments, the molar concentrationof the pharmaceutical agent is within 5 fold or 2 fold of the molarconcentration of NaHCO₃. In some embodiments the molar concentration ofthe pharmaceutical agent is about equal to the molar concentration ofNaHCO₃.

In certain embodiments of the invention, the pharmaceutical compositionfurther includes a chemotherapeutic agent. In some embodiments thechemotherapeutic agent is altretamine, asparaginase, BCG, bleomycinsulfate, busulfan, camptothecin, carboplatin, carmusine, chlorambucil,cisplatin, claladribine, 2-chlorodeoxyadenosine, cyclophosphamide,cytarabine, dacarbazine imidazole carboxamide, dactinomycin,daunorubicin-dunomycin, dexamethosone, doxurubicin, etoposide,floxuridine, fluorouracil, fluoxymesterone, flutamide, fludarabine,goserelin, hydroxyurea, idarubicin HCL, ifosfamide, interferon alfa,interferon alfa 2a, interferon alfa 2b, interfereon alfa n3, irinotecan,leucovorin calcium, leuprolide, levamisole, lomustine, megestrol,melphalan, L-sarcosylin, melphalan hydrochloride, MESNA,mechlorethamine, methotrexate, mitomycin, mitoxantrone, mercaptopurine,paclitaxel, plicamycin, prednisone, procarbazine, streptozocin,tamoxifen, 6-thioguanine, thiotepa, topotecan, vinblastine, vincristineor vinorelbine tartrate.

In some embodiments, the invention relates to kits containing thepharmaceutical compositions of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the survival time of VX2 tumor bearing rabbits that hadreceived no treatment (control), 5 mg of 3-BrPA in PBS, 10 mg of 3-BrPAin PBS, or 50 mg of 3-BrPA in PBS.

FIG. 2 shows the survival time of VX2 tumor bearing rabbits that hadreceived no treatment (control), 50 mg of 3-BrPA in 1M NaHCO₃, 500 mg of3-BrPA in 1M NaHCO₃, or 1000 mg of 3-BrPA in 1M NaHCO₃.

FIG. 3 shows the survival time of VX2 tumor bearing rabbits that hadreceived 500 mg of 3-BrPA in a pH 4 Sodium Bicarbonate buffered solution(Group 1), 500 mg of 3-BrPA in PBS (Group 2), 500 mg of 30BrPA in a pH 7Sodium Bicarbonate buffered solution (Group 3), or no treatment (Group4).

FIG. 4A shows the ATP levels (cell viability) of Hep3B, HepG2, SK-Hep1and VX2 liver cancer cells when treated with various concentrations of3-BrPA for 3 hours.

FIG. 4B shows the ATP levels (cell viability) of Hep3B, HepG2, SK-Hep1and VX2 liver cancer cells when treated with various concentrations of3-BrPA for 24 hours.

FIG. 4C shows the ATP levels (cell viability) of Hep3B, HepG2, SK-Hep1and VX2 liver cancer cells when treated with various concentrations of3-BrPA for 48 hours.

FIG. 5 shows the flow cytometry analysis of 7-AAD and Anexin V levels ofHep3B, HepG2, SK-Hep1 and VX2 liver cancer cells when untreated(Control) or treated with 200 μM 3-BrPA for 2 hours (Treated).

FIG. 6 shows the percent of Hep3B, HepG2, SK-Hep1 and VX2 liver cancercells undergoing apoptosis following treatment with 0 μM (−), 100 μM (+)or 200 μM (++) 3-BrPA.

FIG. 7 shows fluorescent and light micrographs of Annexin V stainedHep3B, HepG2, SK-Hep1 and VX2 liver cancer cells.

FIG. 8 shows a western blot of activated Caspase 3 using lysates fromHep3B, HepG2, SK-Hep1 and VX2 liver cancer cells treated with 0 μM, 100μM, 150 μM or 200 μM 3-BrPA.

FIG. 9 shows the cell viability of H1299 lung cancer cells when treateduntreated (control) or treated with various concentrations of 3-BrPA.

FIG. 10 shows the ATP levels (cell viability) of H1299 lung cancer cellswhen treated untreated (control) or treated with various concentrationsof 3-BrPA.

FIG. 11 shows the viability of MCF7 breast cancer cells when treateduntreated (control) or treated with various concentrations of 3-BrPA.

FIG. 12 shows the ATP levels (cell viability) of MCF7 breast cancercells when treated untreated (control) or treated with variousconcentrations of 3-BrPA.

FIG. 13 shows the ATP levels (cell viability) of MDA MB 231 breastcancer cells when treated untreated (control) or treated with variousconcentrations of 3-BrPA.

FIG. 14 shows the tumor size and mitotic index in pancreatic tumorstaken from orthotopic xenograft pancreatic cancer model mice after 50days of 3-BrPA treatment.

FIG. 15 shows total amount, volume and concentration of 3-BrPAadministered to nude mice to determine the effect of 3-BrPAconcentration on the tolerability of oral 3-BrPA administration.

DETAILED DESCRIPTION I. Definitions

The present invention provides novel methods and compositions for theadministration of 3-halopyruvate as an anti-cancer agent. In order forthe present invention to be more readily understood, certain terms andphrases are defined below and throughout the specification.

“Therapeutic agent” or “pharmaceutical agent” refers to an agent capableof having a desired biological effect on a host. Chemotherapeutic andgenotoxic agents are examples of therapeutic agents that are generallyknown to be chemical in origin, as opposed to biological, or cause atherapeutic effect by a particular mechanism of action, respectively.Examples of therapeutic agents of biological origin include growthfactors, hormones, and cytokines. A variety of therapeutic agents isknown in the art and may be identified by their effects. Certaintherapeutic agents are capable of regulating red cell proliferation anddifferentiation. Examples include chemotherapeutic nucleotides, drugs,hormones, non-specific (e.g. non-antibody) proteins, oligonucleotides(e.g., antisense oligonucleotides that bind to a target nucleic acidsequence (e.g., mRNA sequence)), peptides, and peptidomimetics.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“Therapeutic effect” refers to a local or systemic effect in animals,particularly mammals, and more particularly humans, caused by apharmacologically active substance. The term thus means any substanceintended for use in the diagnosis, cure, mitigation, treatment orprevention of disease or in the enhancement of desirable physical ormental development and conditions in an animal or human. The phrase“therapeutically-effective amount” means that amount of such a substancethat produces some desired local or systemic effect at a reasonablebenefit/risk ratio applicable to any treatment. In certain embodiments,a therapeutically effective amount of a compound will depend on itstherapeutic index, solubility, and the like. For example, certaincompounds discovered by the methods of the present invention may beadministered in a sufficient amount to produce a reasonable benefit/riskratio applicable to such treatment.

“Modulation” refers to up regulation (i.e., activation or stimulation),down regulation (i.e., inhibition or suppression) of a response, or thetwo in combination or apart.

The phrases “therapeutically-effective amount” and “effective amount” asused herein means that amount of a compound, material, or compositioncomprising a compound of the present invention which is effective forproducing some desired therapeutic effect in at least a sub-populationof cells in an animal at a reasonable benefit/risk ratio applicable toany medical treatment.

As used herein, the term “subject” means a human or non-human animalselected for treatment or therapy.

As used herein, the term “subject suspected of having” means a subjectexhibiting one or more clinical indicators of a disease or condition. Incertain embodiments, the disease or condition is cancer. In certainembodiments, the cancer is leukemia or lymphoma.

As used herein, the term “subject in need thereof” means a subjectidentified as in need of a therapy or treatment.

“Treating” a disease in a subject or “treating” a subject having adisease refers to subjecting the subject to a pharmaceutical treatment,e.g., the administration of a drug, such that at least one symptom ofthe disease is decreased or prevented from worsening.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

“Pharmaceutically-acceptable salts” refers to the relatively non-toxic,inorganic and organic salts of compounds.

The term “3-bromopyruvate” or “3-BrPA” as used herein refers to3-bromopyruvate, analogs and derivatives of 3-bromopyruvate, prodrugs of3-bromopyruvate, metabolites of 3-bromopyruvate and salts thereof.

As used herein, the term “cancer” includes, but is not limited to, solidtumors and blood borne tumors. The term cancer includes diseases of theskin, tissues, organs, bone, cartilage, blood and vessels. The term“cancer” further encompasses primary and metastatic cancers.

As used herein, the term “administering” means providing apharmaceutical agent or composition to a subject, and includes, but isnot limited to, administering by a medical professional andself-administering.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered, peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

2. Selective Inhibitors of ATP Production

Some embodiments of the invention relate to the use of 3-halopyruvateand related compounds in the treatment of cancer. In some embodimentsthe 3-halopyruvate is 3-bromopyruvate.

In one aspect, the invention provides selective inhibitors of ATPproduction represented by the general formula:

wherein X represents a halide, a sulfonate, a carboxylate, an alkoxide,or an amine oxide. In certain embodiments, X is a halide selected fromthe group consisting of: fluoride, bromide, chloride, and iodide. In oneembodiment, the inhibitor is a 3-halopyruvate. In certain otherembodiments, the 3-halopyruvate is selected from the group consistingof: 3-fluoropyruvate, 3-chloropyruvate, 3-bromopyruvate and3-iodopyruvate. In one embodiment, the 3-halopyruvate is3-bromopyruvate. In other embodiments, X is a sulfonate and may beselected from the group consisting of: triflate, mesylate and tosylate.In yet another embodiment, X is an amine oxide is dimethylamine oxide.In certain embodiments R₁ represents OR, H, N(R″)₂, C1-C6 alkyl, C6-C12aryl, C1-C6 heteroalkyl, or a C6-C12 heteroaryl. Independently, in otherembodiments, R″ represents H, C1-C6 alkyl, or C6-C12 aryl.Independently, in still other embodiments, R represents H, alkali metal,C1-C6 alkyl, C6-C12 aryl or C(O)R′; and R′ represents H, C1-C20 alkyl orC6-C12 aryl.

In a preferred embodiment, the invention further provides inhibitors ofATP production represented by general formula:

X—CH2-CO—COOH,

-   -   wherein X represents a halide, a sulfonate, a carboxylate, an        alkoxide, or an amine oxide. In certain embodiments, X is a        halide and may be selected from the group consisting of:        fluoride, bromide, chloride, and iodide. In one embodiment, the        inhibitor is 3-halopyruvate. In certain embodiments, the        3-halopyruvate is selected from the group consisting of:        3-fluoropyruvate, 3-chloropyruvate, 3-bromopyruvate and        3-iodopyruvate. In one embodiment, the 3-halopyruvate is        3-bromopyruvate. In other embodiments, X is a sulfonate selected        from the group consisting of: triflate, mesylate and tosylate.        In yet another embodiment, X is an amine oxide is dimethylamine        oxide.

Other analogs, derivatives, prodrugs, metabolites and salts thereof of3-bromopyruvate may also be used, provided that these compounds orcompositions have an anticancerous effect that is statistically similarto that of 3-bromopyruvate. When referring herein to a treatment using3-bromopyruvate, it should be understood that the treatment may also beconducted with analogs, derivatives, prodrugs, metabolites and salts of3-bromopyruvate, where applicable.

3. Pharmaceutical Compositions of the Subject Inhibitors

The invention provides pharmaceutical compositions comprising3-bromopyruvate as well as other inhibitor compounds described above. Inone aspect, the present invention provides pharmaceutically acceptablecompositions which comprise a therapeutically-effective amount of one ormore of the compounds described above, formulated together with one ormore pharmaceutically acceptable carriers (additives) and/or diluents.In another aspect the compounds of the invention can be administered assuch or in admixtures with pharmaceutically acceptable carriers and canalso be administered in conjunction with other chemotherapeutic agentsand/or scavenger compounds. Conjunctive therapy thus includessequential, simultaneous and separate, or co-administration of theactive compound, wherein the therapeutic effects of the firstadministered has not entirely disappeared when the subsequent compoundis administered.

In some embodiments the pharmaceutical composition of the invention isformulated so as to have a specific pH. In some embodiments the pH ofthe pharmaceutical composition of the invention is greater than or equalto about pH 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9 or 4.0. In some embodiments the pH of the pharmaceuticalcomposition of the invention is less than or equal to about pH 9.0, 8.0,7.0, 6.5, 6.0, 5.5, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1 or4.0. The pH of the pharmaceutical composition can be determined by anymethod known in the art or calculated based on the chemical propertiesof the molecules that make up the pharmaceutical composition. The pH ofa pharmaceutical composition of the invention can be adjusted to adesired level using any technique known in the art. In some embodimentsa buffer is used to establish and/or maintain a desired pH in apharmaceutical composition of the invention. In some embodiments, adesired pH of the pharmaceutical composition is established by usingSodium Bicarbonate (NaHCO₃). In some embodiments the molar concentrationof NaHCO₃ in the pharmaceutical composition is greater than or equal to1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0,9.0, 10.0, 15, 20, 25, 50 or 100 times the molar concentration of 3-BrPAin the pharmaceutical composition. In some embodiments the molarconcentration of 3-BrPA in the pharmaceutical composition is greaterthan or equal to 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0,6.0, 7.0, 8.0, 9.0, 10.0, 15, 20, 25, 50 or 100 times the molarconcentration of NaHCO₃ in the pharmaceutical composition.

As described in detail below, the pharmaceutical compositions of thepresent invention may be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, e.g., those targeted for buccal,sublingual, and systemic absorption, boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; (3) topical application, for example, asa cream, ointment, or a controlled-release patch or spray applied to theskin; (4) intravaginally or intrarectally, for example, as a pessary,cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8)nasally.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. These salts can be prepared in situin the administration vehicle or the dosage form manufacturing process,or by separately reacting a purified compound of the invention in itsfree base form with a suitable organic or inorganic acid, and isolatingthe salt thus formed during subsequent purification. Representativesalts include the hydrobromide, hydrochloride, sulfate, bisulfate,phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate,laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate,fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate,lactobionate, and laurylsulphonate salts and the like (see, for example,Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. These salts can likewise be prepared in situ in theadministration vehicle or the dosage form manufacturing process, or byseparately reacting the purified compound in its free acid form with asuitable base, such as the hydroxide, carbonate or bicarbonate of apharmaceutically-acceptable metal cation, with ammonia, or with apharmaceutically-acceptable organic primary, secondary or tertiaryamine. Representative alkali or alkaline earth salts include thelithium, sodium, potassium, calcium, magnesium, and aluminum salts andthe like. Representative organic amines useful for the formation of baseaddition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like (see, for example,Berge et al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated and the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,liposomes, micelle forming agents, e.g., bile acids, and polymericcarriers, e.g., polyesters and polyanhydrides; and a compound of thepresent invention. In certain embodiments, an aforementioned formulationrenders orally bioavailable a compound of the present invention.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol, glycerolmonostearate, and non-ionic surfactants; (8) absorbents, such as kaolinand bentonite clay; (9) lubricants, such a talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-shelled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. Compositions of the invention may also beformulated for rapid release, e.g., freeze-dried. They may be sterilizedby, for example, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

Exemplary formulations comprising 3-bromopyruvate are determined basedon various properties including, but not limited to, chemical stabilityat body temperature, functional efficiency time of release, toxicity andoptimal dose.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, administration by injection, infusion orinhalation; topical by lotion or ointment; and rectal by suppositories.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracistemally and topically, as by powders, ointments ordrops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

In certain embodiments, the above-described pharmaceutical compositionscomprise one or more of the inhibitors, a second chemotherapeutic agent,and optionally a pharmaceutically acceptable carrier.

The term chemotherapeutic agent includes, without limitation,platinum-based agents, such as carboplatin and cisplatin; nitrogenmustard alkylating agents; nitrosourea alkylating agents, such ascarmustine (BCNU) and other alkylating agents; antimetabolites, such asmethotrexate; purine analog antimetabolites; pyrimidine analogantimetabolites, such as fluorouracil (5-FU) and gemcitabine; hormonalantineoplastics, such as goserelin, leuprolide, and tamoxifen; naturalantineoplastics, such as taxanes (e.g., docetaxel and paclitaxel),aldesleukin, interleukin-2, etoposide (VP-16), interferon alfa, andtretinoin (ATRA); antibiotic natural antineoplastics, such as bleomycin,dactinomycin, daunorubicin, doxorubicin, and mitomycin; and vincaalkaloid natural antineoplastics, such as vinblastine and vincristine.

Further, the following drugs may also be used in combination with anantineoplastic agent, even if not considered antineoplastic agentsthemselves: dactinomycin; daunorubicin HCl; docetaxel; doxorubicin HCl;epoetin alfa; etoposide (VP-16); ganciclovir sodium; gentamicin sulfate;interferon alfa; leuprolide acetate; meperidine HCl; methadone HCl;ranitidine HCl; vinblastin sulfate; and zidovudine (AZT). For example,fluorouracil has recently been formulated in conjunction withepinephrine and bovine collagen to form a particularly effectivecombination.

Still further, the following listing of amino acids, peptides,polypeptides, proteins, polysaccharides, and other large molecules mayalso be used: interleukins 1 through 18, including mutants andanalogues; interferons or cytokines, such as interferons α, β, and γ;hormones, such as luteinizing hormone releasing hormone (LHRH) andanalogues and, gonadotropin releasing hormone (GnRH); growth factors,such as transforming growth factor-β (TGF-β), fibroblast growth factor(FGF), nerve growth factor (NGF), growth hormone releasing factor(GHRF), epidermal growth factor (EGF), fibroblast growth factorhomologous factor (FGFHF), hepatocyte growth factor (HGF), and insulingrowth factor (IGF); tumor necrosis factor-α & β (TNF-α & β); invasioninhibiting factor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7);somatostatin; thymosin-α-1; γ-globulin; superoxide dismutase (SOD);complement factors; anti-angiogenesis factors; antigenic materials; andpro-drugs.

Chemotherapeutic agents for use with the compositions and methods oftreatment described herein include, but are not limited to alkylatingagents such as thiotepa and cyclosphosphamide; alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomega11; dynemicin, including dynemicin A; bisphosphonates, such asclodronate; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores, aclacinomysins,actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonicacid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes(especially T-2 toxin, verracurin A, roridin A and anguidine); urethan;vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide;thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumcoordination complexes such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); Ifosfamide; mitoxantrone;vincristine; vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11);topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

In another embodiment, the composition of the invention may compriseother biologically active substances, including therapeutic drugs orpro-drugs, for example, other chemotherapeutic agents, scavengercompounds, antibiotics, anti-virals, anti-fungals, anti-inflammatories,vasoconstrictors and anticoagulants, antigens useful for cancer vaccineapplications or corresponding pro-drugs.

Exemplary scavenger compounds include, but are not limited tothiol-containing compounds, such as glutathione, thiourea, and cysteine;alcohols such as mannitol, substituted phenols; quinones, substitutedphenols, aryl amines and nitro compounds.

Various forms of the chemotherapeutic agents and/or other biologicallyactive agents may be used. These include, without limitation, such formsas uncharged molecules, molecular complexes, salts, ethers, esters,amides, and the like, which are biologically active.

4. Therapeutic Methods

The present invention further provides novel therapeutic methods oftreating cancer, including a cancerous tumor comprising administering toa subject, (e.g., a subject in need thereof), an effective amount of3-bromopyruvate or related compound. A subject in need thereof mayinclude, for example, a subject who has been diagnosed with a tumor,including a pre-cancerous tumor, a cancer, or a subject who has beentreated, including subjects that have been refractory to the previoustreatment.

The methods of the present invention may be used to treat any cancerousor pre-cancerous tumor. In certain embodiments, the cancerous tumor hasa highly glycolytic phenotype. For example, highly glycolytic tumors maybe located in a tissue selected from brain, colon, urogenital, lung,renal, prostate, pancreas, liver, esophagus, stomach, hematopoietic,breast, thymus, testis, ovarian, skin, bone marrow and/or uterinetissue. In some embodiments, methods and compositions of the presentinvention may be used to treat any cancer. Cancers that may treated bymethods and compositions of the invention include, but are not limitedto, cancer cells from the bladder, blood, bone, bone marrow, brain,breast, colon, esophagus, gastrointestine, gum, head, kidney, liver,lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue,or uterus. In addition, the cancer may specifically be of the followinghistological type, though it is not limited to these: neoplasm,malignant; carcinoma; carcinoma, undifferentiated; giant and spindlecell carcinoma; small cell carcinoma; papillary carcinoma; squamous cellcarcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrixcarcinoma; transitional cell carcinoma; papillary transitional cellcarcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; and roblastoma,malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; maligmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-Hodgkin's lymphomas; malignant histiocytosis; multiplemyeloma; mast cell sarcoma; immunoproliferative small intestinaldisease; leukemia; lymphoid leukemia; plasma cell leukemia;erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mastcell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairycell leukemia.

The pharmaceutical compositions of the present invention may bedelivered by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually. In certain embodimentsthe pharmaceutical compositions are delivered generally (e.g., via oralor parenteral administration). In certain other embodiments thepharmaceutical compositions are delivered locally through directinjection into a tumor or direct injection into the tumor's blood supply(e.g., arterial or venous blood supply). In some embodiments, thepharmaceutical compositions are delivered by both a general and a localadministration. For example, a subject with a tumor may be treatedthrough direct injection of a composition containing 3-BrPA into thetumor or the tumor's blood supply in combination with oraladministration of a pharmaceutical composition of the present invention.If both local and general administration is used, local administrationcan occur before, concurrently with and/or after general administration.

In certain embodiments, the methods of treatment of the presentinvention, including treating a cancerous or pre-cancerous tumorcomprise administering 3-bromopyruvate in conjunction with a secondagent to the subject. Such methods in certain embodiments compriseadministering pharmaceutical compositions comprising 3-bromopyruvate ora related compound in conjunction with one or more chemotherapeuticagents and/or scavenger compounds, including chemotherapeutic agentsdescribed herein, as well as other agents known in the art. Conjunctivetherapy includes sequential, simultaneous and separate, orco-administration of the active compound in a way that the therapeuticeffects of the first compound administered one have not entirelydisappeared when the subsequent compound is administered. In oneembodiment, the second agent is a chemotherapeutic agent. In anotherembodiment, the second agent is a scavenger compound. In anotherembodiment, the second agent is radiation therapy. In a furtherembodiment, radiation therapy may be administered in addition to3-bromopyruvate and a second agent. In certain embodiments, the secondagent may be co-formulated in the separate pharmaceutical composition.

In some embodiments, the subject pharmaceutical compositions of thepresent invention will incorporate the substance or substances to bedelivered in an amount sufficient to deliver to a patient atherapeutically effective amount of an incorporated therapeutic agent orother material as part of a prophylactic or therapeutic treatment. Thedesired concentration of the active compound in the particle will dependon absorption, inactivation, and excretion rates of the drug as well asthe delivery rate of the compound. It is to be noted that dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions. Typically, dosingwill be determined using techniques known to one skilled in the art.

Dosage may be based on the amount of the composition per kg body weightof the patient. For example, a range of amounts of compositions arecontemplated, including about 0.001, 0.01, 0.1, 0.5, 1, 10, 15, 20, 25,50, 75, 100, 150, 200 or 250 mg or more of such compositions per kg bodyweight of the patient. Other amounts will be known to those of skill inthe art and readily determined.

In certain embodiments, the dosage of the compounds of the inventionwill generally be in the range of about 0.001 mg to about 250 mg per kgbody weight, specifically in the range of about 50 mg to about 200 mgper kg, and more specifically in the range of about 100 mg to about 200mg per kg. In one embodiment, the dosage is in the range of about 150 mgto about 250 mg per kg. In another embodiment, the dosage is about 200mg per kg.

In certain embodiments, the compounds of the invention will beadministered in a pharmaceutical composition. In some embodiments themolar concentration of the compound of the invention in thepharmaceutical composition will be less than or equal to about 2.5 M,2.4 M, 2.3 M, 2.2 M, 2.1 M, 2 M, 1.9 M, 1.8 M, 1.7 M, 1.6 M, 1.5 M, 1.4M, 1.3 M, 1.2 M, 1.1 M, 1 M, 0.9 M, 0.8 M, 0.7 M, 0.6 M, 0.5 M, 0.4 M,0.3 M or 0.2 M. In some embodiments the concentration of the compound ofthe invention will be less than or equal to about 0.10 mg/ml, 0.09mg/ml, 0.08 mg/ml, 0.07 mg/ml, 0.06 mg/ml, 0.05 mg/ml, 0.04 mg/ml, 0.03mg/ml or 0.02 mg/ml.

Alternatively, the dosage of the subject invention may be determined byreference to the plasma concentrations of the composition. For example,the maximum plasma concentration (Cmax) and the area under the plasmaconcentration-time curve from time 0 to infinity (AUC (0-4)) may beused. Dosages for the present invention include those that produce theabove values for Cmax and AUC (0-4) and other dosages resulting inlarger or smaller values for those parameters.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldprescribe and/or administer doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The precise time of administration and amount of any particular compoundthat will yield the most effective treatment in a given patient willdepend upon the activity, pharmacokinetics, and bioavailability of aparticular compound, physiological condition of the patient (includingage, sex, disease type and stage, general physical condition,responsiveness to a given dosage and type of medication), route ofadministration, and the like. The guidelines presented herein may beused to optimize the treatment, e.g., determining the optimum timeand/or amount of administration, which will require no more than routineexperimentation consisting of monitoring the subject and adjusting thedosage and/or timing.

While the subject is being treated, the health of the patient may bemonitored by measuring one or more of the relevant indices atpredetermined times during a 24-hour period. All aspects of thetreatment, including supplements, amounts, times of administration andformulation, may be optimized according to the results of suchmonitoring. The patient may be periodically reevaluated to determine theextent of improvement by measuring the same parameters, the first suchreevaluation typically occurring at the end of four weeks from the onsetof therapy, and subsequent reevaluations occurring every four to eightweeks during therapy and then every three months thereafter. Therapy maycontinue for several months or even years, with a minimum of one monthbeing a typical length of therapy for humans. Adjustments, for example,to the amount(s) of agent administered and to the time of administrationmay be made based on these reevaluations.

Treatment may be initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage may be increased bysmall increments until the optimum therapeutic effect is attained. Inaddition, the combined use 3-bromopyruvate and related compounds and asecond agent, e.g. another chemotherapeutic agent or a scavengercompound, may reduce the required dosage for any individual compoundand/or agent because the onset and duration of effect of the differentcompounds and/or agents may be complimentary.

As described above, 3-bromopyruvate or related compounds may beadministered in combination with radiation therapy. An optimized dose ofradiation therapy may be given to a subject as a daily dose. Optimizeddaily doses of radiation therapy may be, for example, from about 0.25 to0.5 Gy, about 0.5 to 1.0 Gy, about 1.0 to 1.5 Gy, about 1.5 to 2.0 Gy,about 2.0 to 2.5 Gy, and about 2.5 to 3.0 Gy. An exemplary daily dosemay be, for example, from about 2.0 to 3.0 Gy. A higher dose ofradiation may be administered, for example, if a tumor is resistant tolower doses of radiation. High doses of radiation may reach, forexample, 4 Gy. Further, the total dose of radiation administered overthe course of treatment may, for example, range from about 50 to 200 Gy.In an exemplary embodiment, the total dose of radiation administeredover the course of treatment ranges, for example, from about 50 to 80Gy. In certain embodiments, a dose of radiation may be given over a timeinterval of, for example, 1, 2, 3, 4, or 5 minutes, wherein the amountof time is dependent on the dose rate of the radiation source.

In certain embodiments, a daily dose of optimized radiation may beadministered, for example, 4 or 5 days a week, for approximately 4 to 8weeks. In an alternate embodiment, a daily dose of optimized radiationmay be administered daily seven days a week, for approximately 4 to 8weeks. In certain embodiments, a daily dose of radiation may be given asingle dose. Alternately, a daily dose of radiation may given as aplurality of doses. In a further embodiment, the optimized dose ofradiation may be a higher dose of radiation than can be tolerated by thepatient on a daily base. As such, high doses of radiation may beadministered to a patient, but in a less frequent dosing regimen.

The types of radiation that may be used in cancer treatment are wellknown in the art and include electron beams, high-energy photons from alinear accelerator or from radioactive sources such as cobalt or cesium,protons, and neutrons. An exemplary ionizing radiation is an x-rayradiation.

Methods to administer radiation are well known in the art. Exemplarymethods include, but are not limited to, external beam radiation,internal beam radiation, and radiopharmaceuticals. In external beamradiation, a linear accelerator is used to deliver high-energy x-rays tothe area of the body affected by cancer. Since the source of radiationoriginates outside of the body, external beam radiation can be used totreat large areas of the body with a uniform dose of radiation. Internalradiation therapy, also known as brachytherapy, involves delivery of ahigh dose of radiation to a specific site in the body. The two maintypes of internal radiation therapy include interstitial radiation,wherein a source of radiation is placed in the effected tissue, andintracavity radiation, wherein the source of radiation is placed in aninternal body cavity a short distance from the affected area.Radioactive material may also be delivered to tumor cells by attachmentto tumor-specific antibodies. The radioactive material used in internalradiation therapy is typically contained in a small capsule, pellet,wire, tube, or implant. In contrast, radiopharmaceuticals are unsealedsources of radiation that may be given orally, intravenously or directlyinto a body cavity.

Radiation therapy may also include sterotactic surgery or sterotacticradiation therapy, wherein a precise amount of radiation can bedelivered to a small tumor area using a linear accelerator or gammaknife and three dimensional conformal radiation therapy (3DCRT), whichis a computer assisted therapy to map the location of the tumor prior toradiation treatment.

Toxicity and therapeutic efficacy of subject compounds may be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ and the ED₅₀. Compositions thatexhibit large therapeutic indices are preferred. Although compounds thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets the compounds to the desired site in orderto reduce side effects.

The data obtained from the cell culture assays and animal studies may beused in formulating a range of dosage for use in humans. The dosage ofany supplement, or alternatively of any components therein, liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For agents of the present invention, the therapeuticallyeffective dose may be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information may be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

EXEMPLIFICATION

The invention now being generally described will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

Materials and Methods Animal Model

New Zealand White rabbits were used in the study. All rabbits, carriersand recipients, were anesthetized with a mixture of acepromazine (2.5mg/kg; Phoenix, St. Joseph, Mo.) and ketamine hydrochloride (44 mg/kg;Phoenix) administered intramuscularly. The VX2 tumor cell suspension wasfirst injected into the hind legs of carrier rabbits and grown for 2weeks. Resultant tumors were harvested from each carrier and a tumorsuspension was prepared from each harvested tumor by dissection ofviable tumor tissue and aseptic mincing.

For the rabbits that were going to receive the VX2 tumor implanted inthe liver, intravenous access was gained via a marginal ear vein and0.1-0.2 ml (2.5-5 mg) of sodium pentobarbital (Abbott Laboratories,Abbott Park, Ill.) was given periodically to maintain anesthesia. Theabdomen of each recipient rabbit was shaved and disinfected with ethanoland povidine iodine. The liver of the rabbit was exposed by a midlineincision, then an aliquot of the tumor cell suspension (0.2 ml) wasinjected directly using a 21-gauge angiocatheter into the left lobe ofthe liver in order to develop a solitary lesion with adequatesurrounding liver parenchyma. The abdomen was closed in two layers. Thetumor was allowed to grow in the rabbit livers for 7 days. Pain anddistress levels were assessed by daily by monitoring food intake, fecesand urine production and finally checking for signs of guarding orreluctance to move when touched. Animals were euthanized 6 months afterinitiation of the treatment or when they became moribund or showed signsof distress (e.g. irregular behavior, lethargy, or >20% weight loss).

Cells and Reagents

The non-small cell lung cancer (NSCLC) cell line NCI-H1299, the breastcancer cell lines MCF7 and MDA MB 231 and the Hep3B, Hep2G and SK-Hep1cells were obtained from the American Type Culture Collection (ATCC)(Manassas, Va., USA) and maintained as a monolayer culture in Eagle'sminimum essential medium (MEM, Invitrogen, Gaithersburg, Md., USA)supplemented with 10% fetal bovine serum at 37 C and 5% CO₂. Alltreatments were done in triplicate samples on 96-well plates. VX2 cellswere maintained as described in Geschwind et al., Cancer Res.,62:3909-3913 (2002).

ATP Dependant Luminescence Cell Viability Assay

Cytotoxicity of 3-BrPA was evaluated by the IC₅₀ values, representingthe drug concentration which depletes ATP by 50% compared to non-treatedcontrol. On day 1, cells were seeded into 96-well plates in a volume of100 μl per well. On day 2, the medium of all wells was removed and analiquot of 100 μl of media having serial dilutions of 3-BrPA in PBS,NaHCO₃, NaOH, KOH and KHCO₃ was added to the appropriate well. After 24h of drug exposure cytotoxic effects of 3-BrPA were monitored bycellular ATP level using CellTiter-Glo™ assay reagents from Promega Corp(Madison, Wis.). Briefly, 100 μl assay reagent was added to each welland mixed at room temperature for 2 min to develop luminescent signal.The luminescence intensity (cps) from each sample was determined with aspectrometer (Model Victor III, Perkin Elmer, Mass.). Relativeluminescence was calculated in proportion to the numbers of living cellsper sample well. The results are expressed as the mean of threeindependent experiments. The intracellular ATP contents were calculatedand normalized by equal cell number and expressed as the percentage ofthe control cells. The IC₅₀ values were estimated graphically fromdose-response plots.

Apoptosis Assays

The apoptosis of 3-BrPA treated cells was evaluated using Annexin Vstaining and the formation of active caspase-3. Cells treated with3-BrPA were stained with anti-Annexin V and analyzed by flow cytometryas well as fluorescent microscopy. Flow cytometry was performed using aBecton-Dickinson FACSCalibur. The gating was done based on unstainedcontrol cells, Annexin V-PE stained and 7-AAD stained control cells. Forfluorescent microscopy, Annexin V staining was performed using Annexin Vstaining Microscopy kit (BD Biosciences, USA) according to the protocolrecommended by the manufacturer. In brief, cells grown in chamberedslides were treated with either 3BrPA or the vehicle, washed once withice-cold PBS followed by two washes with binding buffer, at the end ofthe treatment period. Cells were then incubated with the FITC-conjugatedAnnexin V fluorescent antibody for 15 min followed by a gentle wash. Thecells were kept in binding buffer and images were observed capturedusing a Zeiss Axiovert 200 Microscope (Carl Zeiss Microimaging Inc.,USA). Active caspase-3, an apoptotic marker, was identified by westernblot analysis. Lysates prepared from equal numbers of cells treated withdifferent concentrations of 3BrPA were subjected to SDS-PAGEelectrophoresis and immunoblotting against active (cleaved) caspase-3.

Example 1 Oral Administration of 3-BrPA Dissolved in PBS

Preclinical studies of 3-BrPA as a cancer agent have utilized directintra-arterial injection into the liver as the drug delivery method.Though intra-arterial injection reduces the risk of systematic toxicity,it is a technically challenging delivery method that is not applicableto all forms of cancer. Formulations of 3-BrPA for oral administrationwere therefore examined.

Oral administration of 3-BrPA dissolved in PBS was examined using theVX2 tumor model, an animal model for hepatocellular carcinoma (describedabove). VX2 tumors were allowed to grow the livers of 12 New ZealandWhite rabbits for 10 days. Tumor bearing rabbits were divided in 4groups. Group 1 (n=3) received 5 mg of 3-BrPA in 2.75 ml of PhosphateBuffered Saline (PBS) orally once a day, group 2 (n=3) received 10 mg of3-BrPA in 2.75 ml of PBS orally once a day, group 3 (n=3) received 50 mgof 3-BrPA in 2.75 ml of PBS orally once a day and group 4 (control, n=3)received no treatment. Results of the experiment are shown in FIG. 1.Median survival for the animals in group 1 (n=3), receiving 5 mg of3-BrPA in 2.75 ml of PBS, was 66 days. Animals in group 2 (n=3),receiving 10 mg of 3-BrPA in 2.75 ml of PBS, survived for 70 days. Allthe animals in group 3 (n=3), receiving 50 mg of 3-BrPA in 2.75 ml ofPBS expired within 24 hours of oral administration of the first dose.Control animals showed a median survival of 54 days.

Example 2 Oral Administration of 3-BrPA Dissolved in NaHCO₃

Based on the results described in Example 1, it was concluded that 10 mgof 3-BrPA is the maximum tolerated dose (MTD) if dissolved in 2.75 ml ofPBS. In order to enable administration of 3-BrPA in a higher dose, andthus improve survival, the efficacy of 3-BrPA when dissolved in severalalternative buffers was tested in vitro. When dissolved in NaHCO₃,3-BrPA did not show a significantly different in IC₅₀ value asdetermined by an ATP luminescent assay (described above) when comparedto 3-BrPA dissolved in PBS. On the other hand, other buffers tested(NaOH, KOH and KHCO₃), resulted in significantly higher IC₅₀ values.

To test whether buffering 3-BrPA with NaHCO₃ would allow oraladministration of a higher dose of 3-BrPA, the maximum tolerated dose oforally administered 3-BrPA dissolved in NaHCO₃ was determined. As inExample 1, the VX2 tumor model was used, with VX2 tumors grown in thelivers of 12 New Zealand White rabbits for 10 days. Once again, thetumor bearing rabbits were divided in 4 groups. As shown in FIG. 2,median survival for the animals in group 1 (n=3) receiving 50 mg of3-BrPA in 2.75 ml of NaHCO₃ (1M) orally once a day was 60 days. Theanimals in group 2 (n=3) received 500 mg of 3-BrPA in 2.75 ml of NaHCO₃orally once a day survived and had a median survival time of 100 days.All animals in group 3 (n=3) expired within 24 hours after receiving1000 mg of 3-BrPA in 2.75 ml of NaHCO₃ orally. Control animals that didnot receive 3-BrPA had a median survival of 54 days.

Based on these results it was concluded that 500 mg of 3-BrPA is the MTDif buffered with 2.75 ml of 1M NaHCO₃. This dose is a 50 fold increasecompared to the MTD of 3-BrPA in PBS. Significantly, the rabbits thatreceived the MTD of 3-BrPA buffered with 2.75 ml of NaHCO₃ survived 42%longer than rabbits treated with the MTD of 3-BrPA buffered with PBS and79% longer than rabbits that did not receive the 3-BrPA treatment.

The animals treated with the MTD of 3-BrPA buffered with NaHCO₃ died ofaspiration, whereas the other rabbits died due to the tumor itself. Meanliver tumor size in the animals treated with the MTD of 3-BrPA bufferedwith NaHCO₃ was <0.5 cm, whereas mean liver tumor size in all othergroups was >8 cm. The rabbits treated with the MTD of 3-BrPA bufferedwith NaHCO₃ showed no metastases, whereas all other rabbits showedwidespread metastases on pathology.

The effect of pH on the survival of rabbits bearing VX2 liver tumorsthat were treated with the MTD of 3-BrPA buffered with NaHCO3 wasexamined. The VX2 tumor model was once again used, as described above. Atotal of 20 New Zealand White rabbits were included in the study. Allrabbits received VX2 tumor implantation as described above. The tumorwas allowed to grow in the rabbit livers for 7 days. Rabbits weredivided in 4 groups. Group 1 (n=6) received 500 mg of 3-BrPA in 2.75 mlof NaHCO3 (1 M, resulting in pH 4), group 2 (n=2) received 500 mg of3-BrPA in 2.75 ml of PBS (resulting in pH<2), group 3 (n=6) received 500mg of 3-BrPA in 2.75 ml of NaHCO3 (2 M, resulting in pH 7) and group 4(n=6) received no treatment (control group). All drugs were administeredorally once a day.

As shown in FIG. 3, Kaplan Meier analysis of animal survival showed asignificant survival benefit for rabbits treated with 500 mg of 3-BrPAin 2.75 ml of NaHCO3 (1 M, resulting in pH 4) (Group 1) compared torabbits in group 2 (500 mg of 3-BrPA in 2.75 ml of NaHCO3 (0.1 Mresulting in pH<2.0)), group 3 (500 mg of 3-BrPA in 2.75 ml of NaHCO3 (2M, resulting in pH 7)) and group 4 (control group, untreated with3-BrPA). These data underscored the importance and sensitivity of thebuffering conditions used in administration of therapeutic doses of3-BrPA. Unbuffered 3-BrPA, with a pH<2, resulted in immediate death,while buffering conditions that resulted in a pH>6 rendered 3-BrPA lesseffective as a cancer therapeutic.

Example 3 The Effect of 3-BrPA on Liver Cancer Cells

The efficacy of 3-BrPA in the treatment of liver cancer cells wasfurther tested using the cancer cell lines Hep3B, HepG2, SK-Hep1 andVX2. As described above, the cell lines were grown as monolayers in MEM(Invitrogen) supplemented with 10% fetal bovine serum (HyClone) at 37C.° and 5% CO₂, and all treatments were done in triplicate samples on96-well plates.

The effects on cell viability and cellular metabolism of 3-BrPA on Hep3Band VX2 cells were examined using luminescence ATP assays, as describedabove. The level of ATP is an indicator of metabolic activity as well asenergy status of living cells, which decreases as the cells undergoapoptosis or necrosis. Treatment with 3-BrPA resulted in a dose and timedependent decrease in the level of ATP in all four cell lines (FIG.4A-4C). The IC₅₀ value at of 3-BrPA was 130 to 145 μM for HepG2, Hep3Band SK-Hep 1 cells and 60 μM for the Vx-2 cell line, and 125 μM forHepG2, Hep3B and SK-Hep 1 cells and 50 μM for the Vx-2 cell line at 48hours. Thus, increasing the duration of 3BrPA treatment results indecreased IC₅₀ values for the cell lines. These results furthersubstantiate the anti-glycolytic and energy depleting properties of3-BrPA in liver cancer cells.

Flow cytometric analysis of Annexin V-PE and 7-AAD stained Hep3B, HepG2,SK-Hep1 and VX2 cells that were treated with 3-BrPA showed a dosedependent increase in Annexin V positive cells, indicating an elevatedlevels of apoptosis in 3-BrPA treated cells (FIGS. 5 and 6). Comparedwith control, a significant increase the number of apoptotic cells wasobserved in all cell lines tested after 2 hours of treatment with 3-BrPA(FIG. 5).

In order to confirm that the cytotoxic effects of 3BrPA involvescellular apoptotic pathways, Annexin V levels and Caspase-3 activationwas examined in cells treated with 3-BrPA. FIG. 7 shows photomicrographsof Annexin V stained cells either with (Treated) or without (Control)3-BrPA treatment. The elevated levels of Annexin V in the 3-BrPA treatedcells indicate that 2-BrPA induces cell death through apoptosis.Additionally, immunoblotting of cell lysates with activated-Caspase-3specific antibodies confirmed the activation of caspase-3 during 3BrPAmediated cell death (FIG. 8). A dose dependent increase in Caspase-3activation was evident in all four of the cell lines treated with 3BrPA.Overall, these results support the inference that the 3BrPA promotesapoptosis in 3-BrPA treated liver cancer cells and confirms that orallyadministered 3-BrPA may be an effective treatment of liver cancer.

Example 4 The Effect of 3-BrPA on Lung Cancer Cells

The efficacy of 3-BrPA in the treatment of lung cancer cells was testedusing the non-small cell lung cancer (NSCLC) cell line NCI-H1299. Asdescribed above, the cell line was grown as a monolayer in MEM(Invitrogen) supplemented with 10% fetal bovine serum (HyClone) at 37C.° and 5% CO₂, and all treatments were done in triplicate samples on96-well plates.

Trypan blue viability assays revealed a dose and time dependent decreasein viability, after 3-BrPA treatment, in H1299 cells (FIG. 9). At aconcentration of 0.1 mM, more than 80% of the cells died within 3 hours,and 100% cell death was achieved at 0.2 mM. The EC₅₀ of 3-BrPA for H1299cells lies between 0.08 and 0.09 mM. Notably, the EC₅₀ value for 3-BrPAfor H1299 lung cancer cells was lower than the EC₅₀ of 3-BrPA for VX2liver cancer cells, indicating that the lung cancer cell line is moresensitive to 3-BrPA than the liver cancer cell line. Therefore,considering that oral administration of 3-BrPA buffered in NaHCO₃ iseffective in prolonging survival in liver cancer models (Example 2), itis probable that oral administration of 3-BrPA buffered in NaHCO₃ wouldalso be effective in the treatment of lung cancer.

The anti-metabolic effect of 3-BrPA on H1299 cells was examined usingluminescence ATP assays, as described above. The level of ATP is anindicator of metabolic activity as well as energy status of livingcells, which decreases as the cells undergo apoptosis or necrosis.Bioluminescent quantification of ATP in 3-BrPA treated H1299 cellsshowed a dose and time dependent energy (ATP) depletion (cell viability,FIG. 10). This further substantiates the anti-glycolytic and energydepleting properties of 3-BrPA in lung cancer cells, and suggests thatorally administered 3-BrPA may be an effective treatment of lung cancer.

Example 5 The Effect of 3-BrPA on Breast Cancer Cells

The efficacy of 3-BrPA in the treatment of breast cancer cells wastested using the cancer cell lines MCF7 and MDA MB 231. As describedabove, the cell lines were grown as monolayers in MEM (Invitrogen)supplemented with 10% fetal bovine serum (HyClone) at 37 C.° and 5% CO₂,and all treatments were done in triplicate samples on 96-well plates.

Trypan blue viability assays revealed a dose and time dependent decreasein viability, after 3-BrPA treatment, in MCF7 cells (FIG. 11). At aconcentration of 0.05 mM, more than 40% of the cells died within 3 hoursand 100% cell death was achieved at 0.15 mM. The EC₅₀ of 3-BrPA for MCF7cells lies between 0.05 and 0.06 mM. The EC₅₀ value for 3-BrPA for MCF7breast cancer cells was lower than the EC₅₀ of 3-BrPA for VX2 livercancer cells, indicating that the breast cancer cell line is moresensitive to 3-BrPA than the liver cancer cell line. Therefore,considering that oral administration of 3-BrPA buffered in NaHCO₃ iseffective in prolonging survival in liver cancer models (Example 2), itis probable that oral administration of 3-BrPA buffered in NaHCO₃ wouldalso be effective in the treatment of breast cancer.

The anti-metabolic effect of 3-BrPA on MCF7 and MDA MB231 cells wasexamined using luminescence ATP assays, as described above. The level ofATP is an indicator of metabolic activity as well as energy status ofliving cells, which decreases as the cells undergo apoptosis ornecrosis. Bioluminescent quantification of ATP in 3-BrPA treated MCF7cells (FIG. 12) and MDA MB 231 cells (FIG. 13) showed a dose and timedependent energy (ATP) depletion (cell viability). This furthersubstantiates the anti-glycolytic and energy depleting properties of3-BrPA in breast cancer cells, and suggests that orally administered3-BrPA may be an effective treatment of breast cancer.

Example 6 The Effect of 3-BrPA on Pancreatic Cancer

The efficacy of orally administered 3-BrPA in the treatment ofpancreatic cancer was tested using an orthotopic xenograft pancreaticcancer model. Human PANC-10 pancreatic cancer cells were implanted intothe pancreas of NOD/SCID mice and allowed to form 4-5 mm³ sized tumors.Following the establishment of the tumors, tumor-bearing mice wereorally administered 250 μl of a NaHCO₃-buffered solution containingvarying concentrations of 3-BrPA once per day. After 50 days oftreatment, the tumor-bearing mice were sacrificed and tumor growth andcancer cell proliferation was analyzed.

Administration of 3-BrPA reduced the growth of pancreatic cancer in thetumor-bearing mice. At day 50, the tumors in mice that had not beenadministered 3-BrPA (control) had grown to a size of 13-15 mm³, andthere was evidence of local tumor metastases to other parts of thepancreas. The tumors of mice that had been administered NaHCO₃-buffered3-BrPA at a daily dose of 50 mg/kg had grown to be 12-14 mm³ in size,but lacked any signs of local metastases. The tumors of mice that hadbeen administered NaHCO₃-buffered 3-BrPA at a daily dose of 100 mg/kghad grown to be 7-8 mm³ in size, and also lacked any signs of localmetastases. The tumors of mice that had been administeredNaHCO₃-buffered 3-BrPA at a daily dose of 150 mg/kg had grown to be 4-6mm³ in size, and lacked any signs of local metastases. The tumors ofmice that had been administered NaHCO₃-buffered 3-BrPA at a daily doseof 200 mg/kg showed no signs of growth (tumors were 3-5 mm³ in size atday 50), and lacked any signs of local metastases. The results of theseexperiments are summarized in FIG. 14.

The mitotic index of the pancreatic tumors of the 3-BrPA treated micewas analyzed using microscopy in order to determine the how differentdoses of 3-BrPA affected the proliferation of the pancreatic cancercells. The tumors in mice that had not been administered 3-BrPA(control) had a mitotic index of 10 mitotic cells per field. The tumorsof mice that had been administered NaHCO₃-buffered 3-BrPA at a dailydose of 50 mg/kg had a mitotic index of 9-10 mitotic cells per field.The tumors of mice that had been administered NaHCO₃-buffered 3-BrPA ata daily dose of 100 mg/kg had a mitotic index of 7 mitotic cells perfield. The tumors of mice that had been administered NaHCO₃-buffered3-BrPA at a daily dose of 150 mg/kg had a mitotic index of 2-4 mitoticcells per field. The tumors of mice that had been administeredNaHCO₃-buffered 3-BrPA at a daily dose of 200 mg/kg had a mitotic indexof 1-2 mitotic cells per field. The results of these experiments arealso summarized in FIG. 14.

Example 7 The Effect of 3-BrPA Concentration on the Maximum TolerableDose

As demonstrated herein, buffering with NaHCO₃ allows oral administrationof a higher doses of 3-BrPA. To determine the effect of compoundconcentration on the tolerable dose of 3-BrPA, a series of experimentswere performed in nude mice to determine how a change in administeredvolume, and therefore a change 3-BrPA concentration, would influencecompound efficacy and animal survival. For these experiments, 3-BrPa wasadministered at a dose of 200 mg/kg and a pH of 4. FIG. 15 shows thevalues of the administered doses.

All mice in group 1 and 2 (n=3 per group) died within 24 hours afterreceiving 5 mg of 3-BrPA in volumes of 50, and 100 ml, respectively.Mice in groups 3, 4 and 5, receiving 3-BrPA in volumes of 150, 200 and300 ml respectively, did not show any signs of discomfort, and surviveddaily 3-BrPA administration without any apparent ill-effects.

General toxicology studies were conducted on nude mice that were orallyadministered NaHCO₃-buffered 3-BrPA at doses of 0, 100 and 200mg/kg/day. The duration of exposure in this study ranged from a singledose to 3-months of daily dosing. There were no mortalities in thisstudy and animals did not exhibit any treatment related clinicalsymptoms. 3-BrPA treatment had no effect on body weight. Organ weights,gross pathology and histopathology did not show any treatment-relatedchanges in any of the organs examined.

Example 8 3-BrPa does not Bind to Genomic DNA

Many antineoplastic chemotherapeutics are alkylating agents that exerttheir effect by binding to DNA and interfering with cell replication.This results in cumulative toxicities that are dose limiting. Since3-BrPA has been reported to be an alkylating agent, it was determinedwhether 3-BrPA is incorporated into the DNA of 3-BrPA treated cells. Toaccomplish this, genomic DNA was isolated from SK-Hep and Hep3B cellsthat had been exposed to ¹⁴C-3-BrPA. Scintillation counting of theisolated genomic DNA demonstrated no evidence of ¹⁴C incorporation,indicating that 3-BrPA does not bind to genomic DNA.

EQUIVALENTS

The present invention provides, among other things, therapeuticcompositions and methods of treating cancer using 3-bromopyruvate,related compounds, and other selective inhibitors of ATP production.While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification. The appended claims are notintended to claim all such embodiments and variations, and the fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

1. A method of treating cancer in a subject comprising administering tothe subject an effective amount of a pharmaceutical compositioncomprising a pharmaceutical agent represented by the general formula:

wherein, independently of each occurrence: X represents a halide, asulfonate, a carboxylate, an alkoxide, or an amine oxide; R₁ representsOR, H, N(R″)₂, C1-C6 alkyl, C6-C12 aryl, C1-C6 heteroalkyl, or C6-C12heteroaryl; R″ represents H, C1-C6 alkyl, or C6-C12 aryl; R representsH, alkali metal, C1-C6 alkyl, C6-C12 aryl or C(O)R′; and R′ representsH, C1-C20 alkyl or C6-C12 aryl; and wherein the pharmaceuticalcomposition has an acidity of greater than or equal to about pH 2 andless than or equal to about pH
 6. 2. The method of claim 1, wherein thepharmaceutical agent is 3-halopyruvate.
 3. The method of claim 1,wherein the pharmaceutical agent is 3-bromopyruvate.
 4. The method ofclaim 1, wherein the pharmaceutical composition is administered orally.5. The method of claim 1, wherein the pharmaceutical composition furthercomprises NaHCO₃.
 6. The method of claim 5, wherein the molarconcentration of the pharmaceutical agent is within 5 fold of the molarconcentration of NaHCO₃.
 7. The method of claim 5, wherein the molarconcentration of the pharmaceutical agent is within 2 fold of the molarconcentration of NaHCO₃.
 8. The method of claim 5, wherein the molarconcentration of the pharmaceutical agent is about equal to the molarconcentration of NaHCO₃.
 9. The method of claim 1, wherein thepharmaceutical composition has an acidity of greater than or equal toabout pH 3 and less than or equal to about pH
 5. 10. The method of claim1, wherein the pharmaceutical composition has an acidity of about pH 4.11. The method of claim 1, further comprising the administration of achemotherapeutic agent.
 12. The method of claim 11, wherein thechemotherapeutic agent is selected from a group consisting of:altretamine, asparaginase, BCG, bleomycin sulfate, busulfan,camptothecin, carboplatin, carmusine, chlorambucil, cisplatin,claladribine, 2-chlorodeoxyadenosine, cyclophosphamide, cytarabine,dacarbazine imidazole carboxamide, dactinomycin, daunorubicin-dunomycin,dexamethosone, doxurubicin, etoposide, floxuridine, fluorouracil,fluoxymesterone, flutamide, fludarabine, goserelin, hydroxyurea,idarubicin HCL, ifosfamide, interferon alfa, interferon alfa 2a,interferon alfa 2b, interfereon alfa n3, irinotecan, leucovorin calcium,leuprolide, levamisole, lomustine, megestrol, melphalan, L-sarcosylin,melphalan hydrochloride, MESNA, mechlorethamine, methotrexate,mitomycin, mitoxantrone, mercaptopurine, paclitaxel, plicamycin,prednisone, procarbazine, streptozocin, tamoxifen, 6-thioguanine,thiotepa, topotecan, vinblastine, vincristine and vinorelbine tartrate.13. The method of claim 1, wherein the cancer is a solid tumor.
 14. Themethod of claim 1, wherein the cancer is selected from the groupconsisting of: liver cancer, pancreatic cancer, lung cancer and breastcancer.
 15. The method of claim 1, wherein the cancer is liver cancer.16. The method of claim 1, wherein the pharmaceutical agent isadministered at a dose of greater than or equal to about 150 milligramsper kilogram of body weight and less than or equal to about 250milligrams per kilogram of body weight.
 17. The method of claim 1,wherein the concentration of the pharmaceutical agent is less than orequal to about 0.03 milligrams per milliliter.
 18. The method of claim1, wherein the molar concentration of the pharmaceutical agent is about1 M.
 19. A pharmaceutical composition comprising a pharmaceutical agentrepresented in the general formula:

wherein, independently of each occurrence: X represents a halide, asulfonate, a carboxylate, an alkoxide, or an amine oxide; R₁ representsOR, H, N(R″)₂, C1-C6 alkyl, C6-C12 aryl, C1-C6 heteroalkyl, or C6-C12heteroaryl; R″ represents H, C1-C6 alkyl, or C6-C12 aryl; R representsH, alkali metal, C1-C6 alkyl, C6-C12 aryl or C(O)R′; and R′ representsH, C1-C20 alkyl or C6-C12 aryl; and wherein the pharmaceuticalcomposition has an acidity of greater than or equal to about pH 2 andless than or equal to about pH
 6. 20. The pharmaceutical composition ofclaim 19, wherein the pharmaceutical agent is 3-halopyruvate.
 21. Thepharmaceutical composition of claim 19, wherein the pharmaceutical agentis 3-bromopyruvate.
 22. The pharmaceutical composition of claim 19,wherein the pharmaceutical composition is formulated for oraladministration.
 23. The pharmaceutical composition of claim 19, whereinthe pharmaceutical composition further comprises NaHCO3.
 24. Thepharmaceutical composition of claim 23, wherein the molar concentrationof the pharmaceutical agent is within 5 fold of the molar concentrationof NaHCO3.
 25. The pharmaceutical composition of claim 23, wherein themolar concentration of the pharmaceutical agent is within 2 fold of themolar concentration of NaHCO3.
 26. The pharmaceutical composition ofclaim 23, wherein the molar concentration of the pharmaceutical agent isabout equal to the molar concentration of NaHCO3.
 27. The pharmaceuticalcomposition of claim 19, wherein the pharmaceutical composition has anacidity of greater than or equal to about pH 3 and less than or equal toabout pH
 5. 28. The pharmaceutical composition of claim 19, wherein thepharmaceutical composition has an acidity of about pH
 4. 29. Thepharmaceutical composition of claim 19, further comprising achemotherapeutic agent.
 30. The pharmaceutical composition of claim 29,wherein the chemotherapeutic agent is selected from a group consistingof: altretamine, asparaginase, BCG, bleomycin sulfate, busulfan,camptothecin, carboplatin, carmusine, chlorambucil, cisplatin,claladribine, 2-chlorodeoxyadenosine, cyclophosphamide, cytarabine,dacarbazine imidazole carboxamide, dactinomycin, daunorubicin-dunomycin,dexamethosone, doxurubicin, etoposide, floxuridine, fluorouracil,fluoxymesterone, flutamide, fludarabine, goserelin, hydroxyurea,idarubicin HCL, ifosfamide, interferon alfa, interferon alfa 2a,interferon alfa 2b, interfereon alfa n3, irinotecan, leucovorin calcium,leuprolide, levamisole, lomustine, megestrol, melphalan, L-sarcosylin,melphalan hydrochloride, MESNA, mechlorethamine, methotrexate,mitomycin, mitoxantrone, mercaptopurine, paclitaxel, plicamycin,prednisone, procarbazine, streptozocin, tamoxifen, 6-thioguanine,thiotepa, topotecan, vinblastine, vincristine and vinorelbine tartrate.31. A kit comprising the pharmaceutical composition of claim 19.